Nanoimprint lithography formation of functional nanoparticles using dual release layers

ABSTRACT

Functional nanoparticles may be formed using at least one nanoimprint lithography step. In one embodiment, sacrificial material may be patterned on a multilayer substrate including one or more functional layers between removable layers using an imprint lithography process. At least one of the functional layers includes a functional material such as a pharmaceutical composition or imaging agent. The pattern may be further etched into the multilayer substrate. At least a portion of the functional material may then be removed to provide a crown surface exposing pillars. Removing the removable layers releases the pillars from the patterned structure to form functional nanoparticles such as drug or imaging agent carriers.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application claims priority to U.S. provisional applicationNo. 61/410,632 filed Nov. 5, 2010, which is hereby incorporated byreference.

BACKGROUND INFORMATION

Nano-fabrication includes the fabrication of very small structures thathave features on the order of 100 nanometers or smaller. Although wellknown within the integrated circuit industry, nano-fabricationtechniques may be applied in the bio-domain, solar cells industry,battery industry and/or other industries. See, for example, U.S. PatentPublication No. 2007/0031505; U.S. Pat. No. 6,918,946; U.S. Pat. No.7,705,237; Kelly et al., Shape-specific monodisperse nano-molding ofprotein particles, J. Am. Chem. Soc. 2008, vol. 130, pgs. 5437-5439; andCanelas et al., Top-down particles fabrication: control of size andshape for diagnostic imaging and drug delivery, WIREs Nanomedicine andNanobiotechnology, 2009, vol. 1, pgs. 391-404.

Imprint lithography techniques include formation of a relief pattern ina formable layer positioned on a substrate. The substrate may be coupledto a motion stage to obtain a desired positioning to facilitate thepatterning process. The patterning process may use a template spacedapart from the substrate and the formable liquid applied between thetemplate and the substrate. The formable liquid is solidified to formfeatures on the substrate conforming to the shape of the template thatcontacts the formable liquid. After solidification, the template isseparated from the features and the substrate is subjected to additionalprocessing to form functional nanoparticles (e.g., drug deliverydevices, batteries, and the like).

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 illustrates a simplified side view of a lithographic system.

FIG. 2 illustrates a simplified side view of the substrate shown in FIG.1 having a patterned layer positioned thereon.

FIG. 3 illustrates a simplified side view of the substrate shown in FIG.2 having multiple protrusions formed thereon.

FIG. 4 illustrates a simplified side view of a pillar formed by releaseof the protrusions of FIG. 3.

FIGS. 5A-5E illustrate simplified side views of formation of multilayernanoparticles by imprint lithography.

FIGS. 6A-6E illustrate simplified side views of formation of multilayernanoparticles by imprint lithography.

FIGS. 7A-7E illustrate simplified side views of formation of multilayernanoparticles by imprint lithography.

FIG. 8 illustrates a multilayer substrate formed by imprint lithography.

FIG. 9 illustrates a multilayer substrate formed by imprint lithography.

FIG. 10 illustrates a flow chart of an exemplary method of formingmultilayer nanoparticles using imprint lithography.

FIG. 11 is a schematic cross-sectional view of a nanoparticle drugcarrier.

FIG. 12 is a plot showing drug release from nanoparticle drug carriersas a function of time.

FIG. 13 is a schematic cross-sectional view of a coated nanoparticledrug carrier with barrier layers.

FIG. 14 is a plot of release rate over time for a multilayerednanoparticle drug carrier.

FIG. 15 is a schematic cross-sectional view of a nanoparticle drugcarrier with drug-loaded layers of varying thickness.

FIG. 16 is a plot showing drug released over time for a nanoparticledrug carrier shown in FIG. 19.

FIGS. 17A and 17B are schematic perspective views of nanoparticle drugscarrier with drug-loaded layers of similar surface area.

FIG. 18 is a plot showing drug released over time for nanoparticle drugcarriers shown in FIG. 21.

FIGS. 19A and 19B are schematic cross-sectional views of a nanoparticledrug carrier with oppositely charged species in different layers.

DETAILED DESCRIPTION

Referring to the figures, and particularly to FIGS. 1 and 2, illustratedtherein is a lithographic system 10 used to form functional nano- and/ormicroparticles on substrate 12. Substrate 12 may be coupled to substratechuck 14. As illustrated, substrate chuck 14 is a vacuum chuck.Substrate chuck 14 however, may be any chuck including, but not limitedto, vacuum, pin-type, groove-type, electrostatic, electromagnetic,and/or the like. Exemplary chucks are described in U.S. Pat. No.6,873,087, U.S. Pat. No. 7,635,445, U.S. Patent Publication No.2006-0172031, U.S. Pat. No. 7,636,999, and U.S. Pat. No. 7,635,263, allof which are hereby incorporated by reference herein in their entirety.

Substrate 12 and substrate chuck 14 may be further supported by stage16. Stage 16 may provide rotational and/or translational motion inrelation to the x, y and z axes. Stage 16, substrate 12, and substratechuck 14 may also be positioned on a base (not shown).

Spaced-apart from substrate 12 is template 18. Template 18 may includemesa 20 extending therefrom towards substrate 12, with mesa 20 having apatterning surface 22 thereon. Further, mesa 20 may be referred to asmold 20. Alternatively, template 18 may be formed without mesa 20.

Template 18 and/or mold 20 may be formed from such materials including,but not limited to, fused-silica, quartz, silicon, organic polymers,siloxane polymers, borosilicate glass, fluorocarbon polymers, metal,hardened sapphire, and/or the like. As illustrated, patterning surface22 comprises features defined by a plurality of spaced-apart recesses 24and/or protrusions 26, though embodiments are not limited to such aconfiguration. For example, patterning surface 22 may be substantiallyflat. Generally, patterning surface 22 may be defined as any originalpattern that forms the basis of a pattern to be formed on substrate 12.Additionally, template 18 may be treated with an anti-adhesion agent(e.g., RelMat®, available from Molecular Imprints, Inc., Austin, Tex. or(tridecafluoro-1,1,2,2-tetrahydrooctyl)trichlorosilane (FOTS)).Exemplary anti-adhesion agents include, but are not limited to thosedescribed in U.S. Pat. No. 6,696,220, which is hereby incorporated byreference herein in its entirety.

Template 18 may be coupled to chuck 28. Chuck 28 may be configured as,but not limited to, vacuum, pin-type, groove-type, electrostatic,electromagnetic, and/or other similar chuck types. Exemplary chucks arefurther described in U.S. Pat. No. 6,873,087, which is herebyincorporated by reference herein in its entirety. Further, chuck 28 maybe coupled to imprint head 30 such that chuck 28 and/or imprint head 30may be configured to facilitate movement of template 18. Additionally,chuck 28 may be configured to adjust and/or vary the structure oftemplate 18 prior to imprinting, during imprinting, and/or subsequent toimprinting (e.g. during separation).

System 10 may further include fluid dispense system 32. Fluid dispensesystem 32 may be used to deposit functional material 34 a on substrate12. Functional material 34 a may include biocompatible materials (e.g.,polyethylene glycol), pharmaceutical compositions with one or moreactive ingredients (e.g., one or more drugs), imaging agents, materialsused in solar cells (e.g., n-type material, p-type material) orbatteries, or other functional materials that demonstrate desirableproperties in nanoparticle form.

Functional material 34 a may be positioned on substrate 12 usingtechniques such as drop dispense, spin-coating, dip coating, spraycoating, chemical vapor deposition (CVD), physical vapor deposition(PVD), thin film deposition, thick film deposition, and/or the like. Itshould be noted that the positioning of functional material 34 onsubstrate 12 may be configured to limit the amount of waste. Forexample, use of drop dispense in positioning of functional material 34on substrate 12, as compared to spin-coating and the like, may limit theamount of non-useable fluid during formation of functionalnanoparticles.

Substrate 12 may include a removable layer 50. Removable layer 50 mayfacilitate separation of solidified functional material 34 a fromsubstrate 12 as described herein. Examples of materials for use inremovable layer 50 may include, but are not limited to PVA, PMMA,gelatin, and the like.

Referring to FIGS. 1 and 2, system 10 may further comprisesolidification source 38 (e.g., energy source) coupled to direct amedium 40 (e.g., energy) along path 42 to solidify functional material34 a. Imprint head 30 and stage 16 may be configured to positiontemplate 18 and/or substrate 12 in superposition with path 42. System 10may be regulated by processor 54 in communication with stage 16, imprinthead 30, fluid dispense system 32 and/or source 38, and may operate on acomputer readable program stored in memory 56.

Either imprint head 30, stage 16, or both may vary a distance betweenmold 20 and substrate 12 to define a desired volume therebetween that isfilled by functional material 34 a. For example, imprint head 30 mayapply a force to template 18 such that mold 20 contacts functionalmaterial 34 a. After the desired volume is filled with functionalmaterial 34 a, source 38 may produce medium 40, e.g. UV radiation,causing functional material 34 a to solidify and/or cross-linkconforming to a shape of surface 44 of substrate 12 and patterningsurface 22, defining patterned layer 46 on substrate 12. Patterned layer46 may comprise a residual layer 48 and/or features (e.g., protrusions47 and recessions 49). Protrusions 47 may have a thickness t₁ andresidual layer 48 may have a thickness t₂.

Referring to FIGS. 2 and 3, after solidification, patterned layer 46 maybe subjected to further processing to clean patterned layer 46 and/orfurther separate protrusions 47 to form pillars or nanoparticles 52. Forexample, patterned layer 46 may be subjected to an oxygen plasmaetching. Etching may remove at least a portion (e.g., some orsubstantially all) of residual layer 48. FIG. 3 shows protrusions 47 onremovable layer 50 after removal of substantially all of residual layer48.

Referring to FIGS. 3 and 4, release of protrusions 47 from substrate 12may form pillars 52. For example, substrate 12 may be subjected to asolution that includes, but is not limited to, water (e.g., de-ionizedwater), organic solvents (e.g., DMSO), inorganic acids (e.g., diluteHF), basic solutions, and/or the like. The solution may releaseprotrusions 47 from substrate 12 to form pillars 52 having a thicknesst₃.

Etching of protrusion 47 subsequent to solidification of functionalmaterial 34 a may distort the configuration of protrusion 47 such thatthickness t₂ of protrusion 47 is substantially different from thicknesst₃ of the resulting pillar 52. The amount of degradation of shape maylimit the accuracy and/or precision of dimensionality when formingpillars 52. Such distortion may be disadvantageous depending on thedesign consideration for the pillar 52. For example, when pillars 52 arefunctional nanoparticles used as drug delivery devices (e.g.,nanoparticle drug carriers), geo-targeting of destinations for pillar 52within a body (e.g., human, animal, and/or the like) may be misdirectedby alterations and/or distortion in shape.

Separation of template 18 from patterned layer 46 may also causeseparation defects in pillars 52. Although release layers such as FOTSor RelMat®, and the like, may be provided on substrate 12, template 18,or both, the surface area of patterned layer 46 coupled to template 18prior to separation may exceed the surface area of patterned layer 46coupled to substrate 12. Materiality of release layers and/or functionalmaterial 34 in combination with the dynamics of the surface area mayprovide separation defects in pillars 52.

U.S. Patent Application Publication No. US 2011/0049096, incorporatedherein by reference in its entirety, describes processes to minimizedegradation and separation distortion in the formation of functionalnanoparticles involving deposition of functional material withinrecessions formed in a removable layer. Functional nanoparticles formedthrough nanoimprint lithography processes described herein may includeone or more different layers disposed between a first removable layerand a second removable layer. Nanoparticles including two or morepharmaceutical compositions in such a layered structure may be used forcontrolled release of multi-drugs selected to target specific regions ofa subject. For example, multilayer nanoparticles including a combinationof selected pharmaceutical compositions may be designed to provide adesired sequence or rate of release of active ingredients (e.g., in acontrolled release manner), thereby enhancing treatment efficacy. Theprocesses described herein provide for the formation of functionalnanoparticles with controlled accuracy and precision and with minimizeddegradation and/or distortion. Such control extends both to theformation of the nanoparticles as a whole as well as to the formation ofdifferent layers that may make up the nanoparticles. For example, wherenanoparticles are formed having at least two functional layers, themultilayer nanoparticle can be more accurately formed than in otherprocesses, with the interfaces between adjacent layers beingsubstantially planar. This allows for more precise control of thevolume, and therefore the functional material (e.g., drug loading) ofeach layer of the nanoparticle, which in turn can provide more precisecontrol of e.g. release kinetics.

FIGS. 5A-5E illustrate various stages in nanoimprint lithographyprocessing of a multilayer substrate to form multilayer nanoparticles.Referring to FIG. 5A, multilayer substrate 80 includes base layer 82,removable layer 84, functional layers 86 a, 86 b, and 86 c (formed fromfunctional materials 86 a′, 86 b′, and 86 c′, respectively), removablelayer 88, and optional adhesion layer 90.

Functional materials 86 a′, 86 b′, and 86 c′ may have uses within thebio-domain, the solar cell industry, the battery industry, and otherareas in which functional nanoparticles are advantageous. For example,functional materials 86 a′, 86 b′, and 86 c′ may include, but are notlimited to, one or more biocompatible polymers, solar cell materials,polymerizable materials, and the like. Solar cell materials include, forexample, n-type material and p-type material. Biocompatible polymersinclude synthetic biocompatible polymers (including, e.g., poly(ethyleneglycol) (PEG), poly(ethylene glycol) acrylate (PEGA), poly(ethyleneglycol) diacrylate (PEGDA), poly(ethylene glycol) methacrylate (PEGMA),poly(ethylene glycol) dimethacrylate (PEGDMA), poly(lactic acid) (PLA),PLA-PEG copolymer, poly(glycolic acid) (PGA), poly(lactide-co-glycolide)copolymer (PLGA), PLGA-PEG copolymer, PEG-PLA-PEG, and P(NIPAAm-co-EMA)(copolymer of Poly(N-isopropyl acrylamide) and poly(ethylmethacrylate))), natural biocompatible polymers (including e.g.,chitosan, dextran, dextran sulfate, agarose, polylysine, pectin, fibrin,carboxymethyl chitin, collagen, and gelatin), or any combination ofsynthetic and natural biocompatible polymers (including, e.g.,chitosan-PEG, collagen-acrylate, alginate-acrylate, P(PEG-co-peptides),alginate-g-(PEO-PPO-PEO), and P(PLGA-co-serine). A biocompatible polymeror a mixture thereof can be used as a drug carrier solution forimprinting. Additional examples of biocompatible polymers includealginate (natural biocompatible polymer) and poly (vinyl alcohol), poly(ethylenoxide), poly (ethyleneimine), poly (vinyl pyrrolidone), andpoly-N-isopropylacrylamide (synthetic biocompatible polymers). Otherexamples of biocompatible polymers are described in Hamidi et al.,Hydrogel nanoparticles in drug delivery, Advanced Drug Delivery Reviews,vol. 60 (2008), pp. 1638-1649, Hans et al, Biodegradable nanoparticlesfor drug delivery and targeting, Current Opinion in Solid State andMaterials Science, 2002. 6(4): p. 319-327, and Hughes, G. A.,Nanostructure-mediated drug delivery, Nanomedicine: nanotechnology,biology, and medicine, 2005. 1(1): p. 22-30, each of which is herebyincorporated by reference herein in its entirety.

Representative drugs or pharmaceutical compositions or therapeuticagents that can be incorporated into functional materials that arecomprised of such biocompatible polymers include, e.g., doxorubicin,isradipine, paclitaxel, estrogen, insulin, cisplatin, siRNA. Otherexamples of drugs, pharmaceutical compositions and therapeutic agentsare described in Hamidi et al., Hydrogel nanoparticles in drug delivery,Advanced Drug Delivery Reviews, vol. 60 (2008), pp. 1638-1649,incorporated herein by reference in its entirety. Imaging agents canalso be incorporated into the functional materials. Resultant functionalnanoparticles can be used for diagnostic imaging purposes, including butnot limited to fluorescent imaging and magnetic resonance imaging (MRI).Such imaging agents can include e.g. fluorescing molecules orsuperparamagnetic iron oxides (SPIOs). Examples of such imaging agentsare described in Brigger et al., Nanoparticles in cancer therapy anddiagnosis, Advanced Drug Delivery Reviews, 2002. 54(5): p. 631-651.

The multilayer substrate 80 may be formed as further described herein.Base layer 82 may be similar to substrate 12 described in relation toFIG. 1. Base layer 82 may be formed of materials including, but notlimited to, fused-silica, quartz, silicon, organic polymers, siloxanepolymers, borosilicate glass, fluorocarbon polymers, metal, hardenedsapphire, and/or the like. Removable layer 84 may be positioned adjacentto base layer 82, and may have properties similar to those of removablelayer 50 described in relation to FIG. 3. For example, removable layer84 may dissolve when subjected to a solution including, but not limitedto, water (e.g., de-ionized water), organic solvents (e.g., DMSO),inorganic acids (e.g., dilute HF), basic solutions, and/or the like.

As shown in FIG. 5A, multilayer substrate 80 includes three functionallayers, however, a multilayer substrate may include any number offunctional layers (e.g., one functional layer, two functional layers,four functional layers, five functional layers, etc.). Patterned layer92, with features 94, may be formed of sacrificial material on removablelayer 88 or adhesion layer 90, such sacrificial material for formingpatterned layer 92 may be formed of materials including, but not limitedto, a polymerizable fluid comprising a monomer mixture as described inU.S. Pat. No. 7,157,036 and/or U.S. Patent Publication No. 2005/0187339,both of which are hereby incorporated by reference herein in theirentirety. Patterned layer 92 may be formed on multilayer substrate 80using an imprint lithography template such as described in relation tothe system 10 and processes described in FIGS. 1 and 2. It should benoted that patterned layer 92 may be formed by other nano-lithographytechniques including, but not limited to, optical lithography, x-raylithography, extreme ultraviolet lithography, scanning probelithography, atomic force microscopic nanolithography, magnetolithography, and/or the like. Base layer 82 may be formed of materialssuch as fused-silica, quartz, silicon, organic polymers, siloxanepolymers, borosilicate glass, fluorocarbon polymers, metal, hardenedsapphire, or the like. In some cases, removable layer 84 may include apolymer such as, for example, poly(methyl methacrylate) (PMMA) orpoly(vinyl alcohol) (PVA). Removable layer 84 may be applied to baselayer 82 in a process including drop dispense, spin-coating, dipcoating, chemical vapor deposition (CVD), physical vapor deposition(PVD), thin film deposition, thick film deposition, or the like.Patterned layer 92, with features 94, may be formed from a polymerizablematerial such as MonoMat®, available from Molecular Imprints, Inc.

Functional materials 86 a′, 86 b′, and 86 c′ used to form functionallayers 86 a, 86 b, and 86 c, respectively, may each include one or moreactive ingredients and a binder material. The binder material mayinclude, for example, polyethylene glycol diacrylate (PEGDA) orpolyethylene glycol dimethacrylate (PEGDMA), a photoinitiator, and asolvent (e.g., water). The active ingredient(s) and binder material foreach layer may be mixed together to form a liquid with a viscositysuitable for the selected method of application. For example, an activeingredient and a binder may be mixed together to form a liquid with aviscosity suitable for inkjet printing (e.g., hundreds of centipoise orless).

To form functional layer 86 a, functional material 86 a′ is disposed onremovable layer 84, and the functional material is solidified.Solidification may include, for example, contact of discrete portions ofthe functional material 86 a′ with a substantially planar (e.g., blank)template to form a substantially continuous layer of the functionalmaterial between the removable layer 84 and the template. Afterformation of a substantially continuous layer of functional material 86a′, the functional material may be solidified (e.g., polymerized)through photo exposure to form solidified functional layer 86 a betweenremovable layer 84 and the template. After solidification of thefunctional material 86 a′, the template may be separated from solidifiedfunctional layer 86 a. Separation of the template from the functionallayer 86 a may be facilitated by pre-treatment of the template toinclude a release agent (e.g., a fluorinated material or a low frictionmaterial such as diamond-like carbon (DLC)) to enhance releaseperformance. In some cases, a template may be formed of materials withinherent release properties, such as polydimethylsiloxane (PDMS) orfluorinated polyether elastomer.

Functional material 86 b′ may be disposed and solidified on functionallayer 86 a in a process similar to that described for the formation offunctional layer 86 a on removable layer 84. Adhesion of functionallayer 86 b to functional layer 86 a may be achieved through bonding ofexposed functional groups (e.g., acrylate or methacrylate groups) at thesurface of functional layer 86 a with functional groups in functionalmaterial 86 b′, respectively, during solidification (e.g.,polymerization) of functional material 86 b′ to form solidifiedfunctional layer 86 b. The bonding may be, for example, covalentbonding. Similarly, functional material 86 c′ may be disposed andsolidified on functional layer 86 b in a process similar to thatdescribed for the formation of functional layer 86 a on removable layer84. Adhesion of functional layer 86 c to functional layer 86 b may beachieved through bonding of exposed functional groups (e.g., acrylate ormethacrylate groups) at the surface of functional layer 86 b withfunctional groups in functional material 86 c′, respectively, duringsolidification (e.g., polymerization) of functional material 86 c′ toform solidified functional layer 86 c.

Removable layer 88, which may have properties similar or dissimilar tothose of removable layer 84 as further detailed herein, may be formed onsolidified functional layer 86 c. Formation of removable layer 88 may besimilar to that described herein for removable layer 84. For example,removable layer 88 may be applied to functional layer 86 c in a processincluding drop dispense, spin-coating, dip coating, chemical vapordeposition (CVD), physical vapor deposition (PVD), thin film deposition,thick film deposition, or the like. In some cases, removable layers 84and 88 have substantially the same composition. In other cases,removable layers 84 and 88 may have different compositions. Removablelayers 84 and 88 are selected to be non-toxic, biocompatible, and inertwith respect the binders and active ingredients in functional layers 86a-86 c. Removable layers 84 and 88 may include, for example, PMMA, PVA,or gelatin.

In some cases, adhesion layer 90 is optionally formed on removable layer88. Adhesion layer 90 may be formed of a composition described in U.S.Pat. No. 7,759,407, which is hereby incorporated by reference herein inits entirety. During processing of patterned layer 92, adhesion layer 90may help minimize separation distortion by adhering patterned layer 92to multilayer substrate 58 during separation of imprint template frompatterned layer 92 a. Patterned layer 92 may be formed on adhesion layer90 or removable layer 88 with a patterned template in a process similarto that described above with respect to FIGS. 1 and 2. Patterned layer92 may be formed, for example, from MonoMat®. Patterned layer 92 may bea pillar tone or hole tone layer. In some cases, a hole tone layer maybe advantageous. Features 94 of patterned layer 92 have a dimension lessthan about 100 nm. For example, a diameter of holes in a hole tone layermay be about 100 nm or less, or about 50 nm or less.

Following the process described with respect to the formation of themultilayer substrate and patterned layer of FIG. 5A, a de-scum etch(e.g., with O₂) can be performed to remove the residual portion ofpatterned layer 92 and underlying portions of adhesive layer 90, ifpresent. As shown in FIG. 5B, the de-scum etch leaves features 94 ofpatterned layer 92 exposed above removable layer 88. With features 94 asa guide, a process such as VUV/O₃, oxygen ashing, reactive ion etching,or argon ion etching may be used to etch through patterned layer 92 intothe multilayer substrate 80.

In patterned layer 92, polymer bond energies may be between about 2 andabout 5 eV. Ions gaining between about 20 and about 2000 eV have theability to alter the polymer surface of patterned layer 92. See, forexample, Egitto, Plasma Etching and Modification of Organic Polymers,Pure and Applied Chemistry, 62:9 (1990) 1699-1708; Kushida et al.,Dry-Etching Durability of Copolymers and Polymer Blends ofVinylnaphthalene or α-Methylstyrene with Methyl Methacrylate, JapaneseJournal of Applied Physics, 34:1:8A (1995) 4234-4238; and Cho et al.,Identification of Hydrophilic Group Formation on Polymer Surface duringAr ⁺ Ion Irradiation in O ₂ Environment, Material Research SocietySymposium Proceedings 438 (1997) 517-532, all of which are herebyincorporated by reference herein in their entirety.

An inert gas, such as argon gas, can be used for dry etching ofpatterned layer 92 in a sputtering process referred to as ion milling oretching. The use of an inert gas may beneficial in avoiding unwantedreactions with functional materials in the functional layers. The energygiven to the Ar⁺ ions can be controlled by controlling the RF bias givento the substrate. Argon can be used as an etchant through two physicaletch processes—argon sputter etching (SE) and argon ion beam etching(IBE). See Koh et al., Surface Modification of Polymer by Ion AssistedReaction in Reactive Gases Environment, Material Research SocietySymposium Proceedings 438 (1997) 505-510, which is hereby incorporatedby reference herein in its entirety.

A high degree of physical etching yields good anisotropy. Selectivitycan be attained by introducing gases like CHF₃, O₂, etc. along with Arto get a physical and chemical etch combination. A pure physical etchprocess may be slower than a combined physical and chemical etchprocess. To increase etch rates, the energy given to the ions may beincreased. Sputter depth profiling shows that sputter etching with Ar⁺ions at 5 keV penetrates several nanometers below the polymer surface.This energy is enough to create homolytic scission of a large number ofC—C and/or C—H bonds forming two radicals with each scission andundergoing subsequent reactions. See Hollander et al., On DepthProfiling of Polymers by Argon Ion Sputtering, Plasma Processes andPolymers, 4 (2007) 773-776, which is hereby incorporated by referenceherein in its entirety.

The homolytic scission may change the composition of patterned layer 92if it is sputtered with Ar⁺ ions at high energy for a long duration.However, the energy, quantity and time of Ar⁺ ions in the plasma can becontrolled to achieve desired results. For example, it has been shownthat Ar⁺ ions with energy of 1 keV did not have significant effect inchanging in surface chemistry (one measure being the contact angle ofwater on the surface which indicates presence of C═O, (C═O)—O, C—O andother hydrophilic groups). Thus, argon ion etching may be used to etch asacrificial patterned layer without adversely affecting (e.g., withoutreacting with) layers in a multilayer substrate. This advantage of argonion etching may be significant, since other etching processes mayinteract adversely (e.g., chemically) with functional layers in themultilayer substrate, for example, altering a pharmaceutical compositionto decrease efficacy.

As shown in FIG. 5C, a pattern of the patterned layer may be transferredthrough multilayer substrate into removable layer 84 to form patternedstructure 98. After the pattern is transferred into the multilayersubstrate 80, a lift-off or release process can be used to formmultilayer nanoparticles with functional layers 86 a-86 c. In somecases, the lift-off process is a one-step process with a single solvent.In other cases, the lift-off process is a two-step process with twodifferent solvents. For example, if removable layers 84 and 88 havedifferent solubilities, removable layer 88 can be dissolved (e.g., bydipping patterned structure 98 in a first solvent) to yield patternedsubstrate 100. As shown in FIG. 5D, after removable layer 88 has beenremoved, functional layers 86 a-86 c remain adhered to removable layer84.

Patterned structure 100 may be further processed to separate multilayernanoparticles from removable layer 84 and substrate 82. For example, themultilayer structure 100 may be further processed, or a second solventcan be used to lift off or dissolve removable layer 84. After removablelayer 84 has been lifted off or dissolved, multilayer nanoparticles 102with functional layers 86 a-86 c, shown in FIG. 5E, may be collectedfrom the second solvent and processed further (e.g., dried). In somecases, a single solvent may be used to dissolve removable layers 84 and88, such that multilayer nanoparticles 102 are formed directly frompatterned structure 98 without forming patterned structure 100

Solvents used to dissolve removable layers 84 and 88 are preferablynon-toxic, biocompatible, and inert with respect the binders and activeingredients in functional layers 86 a-86 c. Solvents that may be usedinclude, for example, water, acetone and/or DMSO. For example, water canbe used as a solvent for a removable layers including, for example PVAor gelatin. Acetone can be used as a solvent for a removable layerincluding PMMA. Removable layer and solvent combinations can be beselected to achieve selective dissolution of the removable layers. Forexample, two removable layers can be formed of PVA and PMMA,respectively. DMSO or water can be used to dissolve the PVA layer, butDMSO or water do not dissolve PMMA. Subsequent treatment with e.g.acetone can then dissolve the PMMA layer. Similarly, the two removablelayers can be formed of PVA and PAA, respectively. Again DMSO can beused to dissolve the PVA layer, but DMSO does not dissolve PAA.Subsequent treatment with water can then dissolve the PAA layer. In somecases, dilute HF is used to dissolve silica-containing layers.Silica-containing layers can be used, for example, as temporary maskinglayers when a substrate is exposed to an oxygen plasma etch.

In some cases, after removable layer 88 is removed and before removablelayer 84 is removed, patterned substrate 100 may be dried. Drying mayinclude, for example, drying at room temperature in an inert atmosphere(e.g., N₂) for about an hour or more (e.g., up to 24 hours, up to 72hours, or longer). Drying patterned substrate 100 before releasing themultilayer nanoparticles from substrate can be advantageous inpost-processing of the resulting multilayer nanoparticles in processesincluding surface loading of the drug with enzymes, with an additionaldrug, or the like or surface charging one or more layers of themultilayer nanoparticles (e.g., surface charging a drug-loaded layer).This drying step provides flexibility to tailor the multilayernanoparticle or nanoparticle drug carrier further to suit in-vitrotesting or in-vivo testing and delivery requirements.

In combination with the above, or as separate processes, resultingnanoparticle surfaces can be further modified prior to release from thesubstrate. For example, the surface of a nanoparticle can befunctionalized with a different functional group (—COO— to —NH2+) toattach subsequent functional molecules (ligands) at these sites, whichcan, for example, enhances the internalization of such nanoparticles bytargeted cells. This may be accomplished by incubation, either insolution or through vapor deposition, with molecules having reactivegroups on one end specific for the available functional group of thefunctional layer polymer(s) and with the other end having the desiredfunctional group. With such approach reactive carboxylic groups on thesurface of nanoparticle can, for example, essentially be replaced withreactive amine groups or vice versa. As another example, nanoparticlescan be exposed to ammonia (NH3) plasma to functionalize the surface ofnanoparticles. Resultant nanoparticles can also be incubated with drugloaded solvent, again either through solution or through vapordeposition, where the drug is absorbed onto on the surface of thenanoparticle after a period of exposure. The surface charge of thenanoparticle can also be modified, i.e., from negative to positive orvice versa, or from a low negative or positive to a high negative orpositive, or vice versa. Such charge modification may improve dispersionstability of the nanoparticle in liquid media, and may play a role inuptake by targeted cells for medial drug delivery and diagnostics. Forexample, adsorption of cationic (Polyethyleneimine, PEI) and anionic(Polyacrlyic acid) polymeric surfactants can modify the surface chargeof the resultant nanoparticles through exposure to such surfactantseither in solution or vapor form. Surface modification can also includeincreasing hydrophillicity or hydrophobicity of the nanoparticlesurface. For example, an increase in hydrophobicity may increase theability of the nanoparticle embed and stick to the hydrophobic layer ofthe bi-lipid cell membrane of animal cells, increasing the chances thecell will internalization of the nanoparticle. Hydrophobicity andhydrophilicity can be modified e.g., through surface energy modificationof polymers using different gas plasma in an RIE etch process. Forexample by using a fluorine gas plasma (CF₄, CHF₃, SF₆) a polymersurface can be made less hydrophilic compared to if it were etched in anoxygen gas plasma (He, Ar, NH₃, O₂). In an example, removable layer 84includes PMMA (which dissolves in acetone) and removable layer 88 (underthe masking layer if present) is PVA (soluble in water). Functionallayers 86 a-86 c are different PEGDA mesh layers, and can independentlybe drug-loaded or non-drug loaded. After water is used to dissolveremovable layer 88, patterned substrate 100 remains. Patterned substrate100 is then exposed to an aqueous solution of polyethyleneimine (PEI,which is cationic and dissolves in water) for a sufficient time to allowat least the negatively charged PEGDA functional layers 86 a-86 b tobecome positively charged. Patterned substrate 100 is then dried, andthe positively charged multilayer nanoparticles 102 are harvested bydissolving removable layer 84 with acetone. Alternatively, negativelycharged multilayer nanoparticles 102 can be formed in a similar processwith different reagents. Charged multilayer nanoparticles are useful forin-vitro and in-vivo studies. For example, for in-vitro studies, cellshave a higher affinity for positively charged nanoparticles thannegatively charged nanoparticles since cell membranes are negativelycharged and have low affinity for internalizing negatively chargedparticles.

In some cases, a hard mask layer may be used in a nanoimprintlithography process to form multilayer nanoparticles. FIG. 6Aillustrates multilayer substrate 80 formed as described with respect toFIG. 5A, with hard mask layer 96 formed on patterned layer 92 by methodsknown in the art with, for example, a silicon-containing material. Hardmask layer 96 may be deposited on patterned layer 92 through a processsuch as spin-coating, CVD, PECVD, imprinting methods, and the like.

Following the process described with respect to the formation of themultilayer substrate 80, patterned layer 92, and hard mask layer 96, asputter or dry etch method may be used to remove at least a portion ofthe hard mask layer 96, exposing portions (e.g. features 94) of thepatterned layer 92, as shown in FIG. 6B. Following exposure of features94, a process such as VUV/O₃, oxygen ashing, reactive ion etching, orargon etching may be used to etch through the multilayer substrate 80 toremovable layer 84 to form patterned structure 100, as shown in FIG. 6C.

After the pattern is transferred into the multilayer substrate 80, aone- or two-step lift-off process similar to that described with respectto FIGS. 5C-5E can be used to form multilayer nanoparticles 102 withfunctional layers 86 a-86 c. In some cases, patterned structure 100,shown in FIG. 6D, may be formed in a two-step lift-off process to formmultilayer nanoparticles 102 shown in FIG. 6E. For a one-step lift-offprocess, multilayer nanoparticles 102 may be formed directly frompatterned structure 98 shown in FIG. 6C.

FIG. 7A illustrates a multilayer substrate 104 having base layer 82,removable layer 84, four functional layers 86 a-86 d, and removablelayer 88. Each functional layer 86 a-86 d can be formed from apharmaceutical composition including a binder and one or more activeingredients. Hard mask layer 96 is adjacent to removable layer 88.Adhesion layer 90 is adjacent to hard mask layer 96. Patterned layer 92is formed on adhesion layer 90 with a hole tone template. Multilayersubstrate 104 may be formed in a process similar to that described formultilayer substrate 80 with respect to FIG. 5A. In an example, baselayer 82 is silicon, removable layer 84 is PVA, removable layer 88 isPVA, hard mask layer 96 is silicon oxide (SiO_(x)), adhesion layer 90 isValMat® (available from Molecular Imprints, Inc.), and patterned layer92 is PEGDA.

Multilayer substrate 104 may be processed to form multilayernanoparticles suitable, for example, as controlled release medications.Processing of multilayer substrate 104 is illustrated in FIGS. 6B-6D.Referring to FIG. 6B, a portion (e.g., the residual portion) ofpatterned layer 92 is etched to expose hard mask layer 96 betweenfeatures 94 of the patterned layer. With portions of hard mask layer 96exposed, multilayer substrate 104 may undergo additional processing toexpose portions of removable layer 84. The additional processing mayinclude an etching step (e.g., dry etching) to yield patterned structure106, as shown in FIG. 6C. The pillars in patterned structure 106 may beetched down to removable layer 88 to yield patterned structure 108,shown in FIG. 6D. A thickness of removable layer 88 can be selected suchthat hard mask layer 96 may be over-etched to ensure substantiallycomplete removal of the hard mask layer, thus removing a portion of theremovable layer 88, and allowing some of the removable layer to remainon patterned structure 108. Patterned structure 108 may be subjected toa lift-off or release process in which removable layers 84 and 88 aredissolved to yield multilayer nanoparticles 102, as shown in FIG. 7E. Inan example, a lift-off process includes immersing the patternedstructure 108 in a solvent (e.g., water) to dissolve removable layers 84and 88, and collecting nanoparticles 102 from the solvent.

FIG. 7E shows a variety of multilayer nanoparticles 102 that may beformed by the process described with respect to FIGS. 7A-7D. Themultilayer nanoparticles 102 may be, for example, substantiallycylindrical or rectangular, or may have an irregular shape. In somecases, a template used to form a patterned layer on a multilayersubstrate may be configured to form hollow pillars rather than solidpillars. That is, a recess in a template may include a protrusion (e.g.,a cylindrical glass rod) such that patterned structures formed in animprint lithography process include hollow multilayer protrusions thatundergo a lift-off process to form multilayer nanoparticles 102 withopenings 110. In an example, a multilayer nanoparticle 102 with opening110 may have an outer diameter on the order of 200 nm and an innerdiameter on the order of 100 nm. Opening 110 provides additional surfacearea for more rapid release of functional materials from the multilayernanoparticle 102.

FIG. 8 illustrates a multilayer substrate 112 having base layer 82,removable layer 84, and five functional layers 86 a-86 e. Eachfunctional layer 86 a-86 e can be formed from a functional material suchas a pharmaceutical composition including a binder and one or moreactive ingredients (e.g., drugs). Hard mask layer 96 is adjacent tofunctional layer 86 e. Adhesion layer 90 is adjacent to hard mask layer96. Patterned layer 92 is formed on adhesion layer 90 with a hole tonetemplate. Multilayer substrate 112 may be formed in a process similar tothat described for multilayer substrate 80 with respect to FIG. 5A. Inan example, base layer 82 is silicon, removable layer 84 is PVA, hardmask layer 96 is silicon oxide (SiO_(x)), adhesion layer 90 is ValMat®(available from Molecular Imprints, Inc.), and patterned layer 92 isPEGDA.

Multilayer substrate 112 may be processed to form multilayernanoparticles. Processing may include, for example, dry etching toremove the hard mask layer, while leaving functional layer 86 e intact,followed by a lift-off or release process to dissolve the removablelayer 84. After removable layer 84 is dissolved, nanoparticles 102 maybe collected.

FIG. 9 illustrates a multilayer substrate 114 having base layer 82,removable layer 84, functional layers 86 a and 86 b, and removable layer88. Patterned layer 92 is formed on removable layer 88, and hard masklayer 96 is formed on removable layer 88. One or more of the functionallayers can be formed from a pharmaceutical composition including abinder and one or more active ingredients. Patterned layer 92 is formedon adhesion layer 90 with a pillar tone template. Multilayer substrate114 may be formed in a process similar to that described for multilayersubstrate 80 with respect to FIG. 5A. In an example, base layer 82 issilicon, removable layer 84 is PVA, removable layer 88 is PVA, patternedlayer 92 is PEGDA, and hard mask layer 96 is silicon oxide (SiO_(x)).

Multilayer nanoparticles 102 may be formed from multilayer substrate 114in a process similar to that described with respect to FIG. 10B. Forexample, portions of the hard mask layer may be etched away to exposefeatures of the patterned layer, and the multilayer substrate 114 may beetched down to removable layer 84 to form multilayer pillars extendingfrom the removable layer. The remaining dry mask material (as well as aportion of removable layer 88, as described with respect to FIG. 11D)may be removed by dry etching. Removable layers 84 and 88 in multilayersubstrate 106 are formed from the same material, and are thus dissolvedby the same solvent. After a lift-off procedure with one solvent torelease multilayer nanoparticles 102 from the base layer 82, thenanoparticles may be collected.

FIG. 10 illustrates a flow chart of a method 120 of forming multilayernanoparticles using imprint lithography. In step 122, a multilayersubstrate is formed. The multilayer substrate includes a base layer, afirst removable layer, two or more functional layers, a second removablelayer, and an optional adhesion layer. One or more or the functionallayers may include a pharmaceutical composition. In step 124, apatterned layer formed of sacrificial material, a hard mask layer, anadhesion layer, or any combination thereof may be formed, in any order,on the multilayer substrate. In step 126, the patterned layer, hard masklayer, and adhesion layer, if present, and the multilayer substrate areetched in one or more processes to form pillars that extend from thebase layer or first removable layer of the multilayer substrate. In step128, one or more lift-off or release processes are performed to separatethe pillars formed in step 126 from the base layer of the substrate.When the first and second removable layers are formed from the samematerial or from materials that have similar solubility properties, asingle lift-off step is needed to release the multilayer nanoparticlesfrom the base layer. When the first and second removable layers areformed from different materials that have different solubilityproperties, two lift-off steps are needed to release the multilayernanoparticles from the base layer. In step 130, the multilayernanoparticles are collected and subjected to further processing asdesired.

There are numerous barriers to drug delivery in human beings. When adrug is delivered through the blood stream it has to overcome barriersincluding cells of the recticuloendothelial system (RES) (e.g., in thespleen, liver, etc.) which are a part of the immune system, renalfiltration (kidney) of blood and biological membrane barriers such asthe plasma membrane, the endosomal membrane and the nuclear membrane ofthe target cell. By creating drug delivery agents at the submicronscale, physical barriers and RES uptake can be avoided thereby allowingefficient travel and uptake of the drug loaded nanoparticles to targetsites in the body. For successful drug delivery the nanoparticle drugsfurther need to overcome resistance at the cellular level to enter thecytoplasm of the cell and reach the ribosome and/or the cell nucleuswhere the drug needs to be released. The nanoparticle drug carriers aretaken up by endosomes of the target cell(s) in order to transport theparticles from the plasma membrane to the lysosome for digestion.Nanoparticles for drug delivery purposes need to break through theendosome membrane or the lysosome membrane to avoid being destroyed bythe lysosome. Once the nanoparticles break through the endosome orlysosome membrane, they enter the cytoplasm. The nanoparticles can thentarget the ribosome or enter the nucleus via the nucleus membrane. Toavoid membrane resistance issues, the nanoparticles loaded with drug canbe engineered to be multifunctional so that it can overcome suchbarriers and release a drug at a pre-defined rate. Multifunctionalitycan be added to nanoparticle drug carriers by adding various agents indifferent layers of a multilayered nanoparticle drug carrier.Nanoparticles can be manufactured in a multilayered fashion for variousembodiments as described below.

For constant or zero order release kinetics, the release rate of drugagents from the nanoparticle carrier is controlled by diffusion of thedrug through the carrier or degradation of the carrier structure/matrix,thereby releasing the drug. It is difficult to control drug releasethrough degradation of carrier structures as these structures swellafter being in the blood and cell medium, thereby increasing the drugdiffusion out of the loose matrices. FIG. 11 shows nanoparticle carrier150 with inner matrix 152 and outer matrices 154. Inner matrix 152includes drug particles 156. Each of outer matrices 154 forms a tighterbarrier layer matrix, compared to the structure of inner matrix 152. Asused herein, a “tighter” matrix generally refers to a more closelypacked polymeric matrix or mesh network. For example, a higher molecularweight polymer with a longer chain length yields a more loosely packedcross-linked structure than a lower molecular weight polymer with ashorter chain length. The presence of outer matrices 154 can curbdiffusion of drug 156 from inner matrix as it degrades in the cell. Thedensity of inner matrix 152 and outer matrices 154 can be selected toachieve a near zero order kinetic profile, thereby supplying drugparticles 156 at a substantially continuous rate over time. FIG. 12 is agraph showing theoretical release kinetics (% drug released vs. time)for drug particles in a substantially homogenous nanoparticle carrier(plot 160) and a nanoparticle carrier with tighter outer matrices 154(plot 162) as shown in FIG. 11.

Near zero-order drug release can also be achieved with a nanoparticlecarrier formed by sandwiching a hydrophobic layer with embedded drugparticles between two hydrophilic layers, between two hydrophobiclayers, or between a hydrophilic layer and a hydrophobic layer, asdescribed in Qui et al., Design and Evaluation of Layered DiffusionalMatrices for Zero-Order Sustained-Release, Journal of ControlledRelease, vol. 51, pp. 123-130, 1998, which is hereby incorporated byreference herein in its entirety.

Nanoparticle drug carriers can be structured in way to release drug atpre-defined time (e.g., programmable release rate) or in a pulsatingfashion (e.g., pulsated release kinetics). As described in Qui et al.,Design of a Core-Shelled Polymer Cylinder for Potential ProgrammableDrug Delivery, International Journal of Pharmaceutics, vol. 219, pp.151-160, 2001, which is hereby incorporated by reference herein in itsentirety, such release kinetics may be favorable for treating certaintypes of diseases. FIG. 13 shows a cross-section of nanoparticle carrier170 with drug-loaded layers 172, 176, and 180 and non-loaded layers 174and 178 stacked to create a repeating unit of drug-loaded and non-loadedlayers. Nanoparticle carrier 170 is partially surrounded by coating 182,such that layers 172, 174, 176, 178, and 180 are released sequentiallyin the direction indicated by the arrows. In some embodiments, coating182 is formed by vapor deposition of a non-toxic hydrophobic materialsuch as PMMA.

The composition of drug-loaded layers 172, 176, and 180 and non-loadedlayers 174 and 178 can be selected to yield a desired release response.For example, if drug-loaded layers 172, 176, and 180 have the samecomposition, the release response shown in FIG. 18 is observed, in whichpeaks 182, 184, and 186 correspond to the release of the same drug fromlayers 172, 176, and 180. In another example, if layers 172, 176, and180 have different compositions, the release response shown in FIG. 14is observed, in which the three peaks 182, 184, and 186 correspond tothe different drugs in layers 172, 176, and 180, respectively. Thisconfiguration is suitable, for example, if the drug in layer 176 needsto be released only after the drug in layer 172 has been delivered.

In certain embodiments, the composition of each drug-loaded layer 172,176, and 180 and non-loaded layer 174 and 178 is selected such thatrelease of the drugs in layers 172, 176, and 180 is triggered byspecific changes in parameters (e.g., temperature, pH, etc.). This canbe achieved by incorporating effective triggering agents into thevarious layers, such that a drug is released selectively in response toa particular stimulus (e.g., the presence of an enzyme or a selected pHor temperature of the environment). In an example, release of a plasmidDNA from a nanoparticle drug carrier is triggered by the presence of anenzyme. See Glangchai et al., Nanoimprint lithography based fabricationof shape-specific enzymatically-triggered smart nanoparticles, Journalof Controlled Release, vol. 125 (2008), pp. 263-272.

Programmed dual drug release can be achieved using a multilayerednanoparticle carrier in which two or more drugs have different initialdrug release times. The different initial drug release times can beselected based on physical shape of the nanoparticle carrier (e.g.,thickness, size) and internal matrix structure (polymer chain length,molecular weight) of each layer, as described in Okuda et al.,Time-programmed Dual Release Formulation by Multilayered Drug-loadedNanofiber Meshes, Journal of Controlled Release, vol. 143, pp. 258-264(2010), which is hereby incorporated by reference herein in itsentirety. FIG. 15 shows a schematic cross-sectional view of a programmeddual drug release nanoparticle carrier 190, with first drug-loaded layer192, first barrier layer 194, second drug-loaded layer 196, and secondbarrier layer 198. The drug in layer 192 is released first. The drug inlayer 196, sandwiched between barrier layers 194 and 198, is releasedlater.

FIG. 16 shows a graph with plots 200 and 202 showing the release overtime of the drugs in first drug-loaded layer 192 and second drug-loadedlayer 196, respectively. The drug in first drug-loaded layer 192 isreleased first. The composition of the barrier layers 194 and 198 can beselected to achieve a desired time difference Δt between the end of therelease of the drug in first drug-loaded layer 192 and the beginning ofthe release of the drug in the second drug-loaded layer 196. Assuming asimilar concentration of the two drugs and other factors, the differencein thickness of layers 192 and 196 may be seen along the y axis as adifference in amount of drug released. The time difference Δt can betailored from being tens of minutes to a few hours. The structure ormatrix of the barrier layers 194 and 198 can be altered (e.g., withrespect to size and/or mesh density) in order to tailor the timedifference Δt between drug release from layers 192 and 196.

FIG. 17A shows nanoparticle drug carrier 210 with drug-loaded layers 212and 214. FIG. 17B shows nanoparticle drug carrier 210 with drug-loadedlayers 212 and 214 separated by barrier layer 216. Barrier layer 216inhibits diffusion of a component (e.g., a drug) in layer 210 fromdiffusing into layer 212 and vice versa. Nanoparticle drug carriers 210are designed such that drug-loaded layers 212 and 214 have substantiallyequal surface area. Since the release of a drug from a drug-loaded layerof a nanoparticle drug carrier is related to the surface area of thedrug-loaded layer exposed to bodily fluids, the rate of release of drugsfrom layers 212 and 214 in a subject is substantially the same. FIG. 18shows a near zero-order rate of release for drugs in nanoparticle drugcarrier 210, with plots 220 and 222 showing substantially the samepercent of drug release over time from drug-loaded layers 212 and 214,respectively,

Once a drug escapes the endosome or lysosome, it enters the cytoplasm.Translocation of molecules from the cytoplasm to the nucleus of the celloccurs through the nuclear pore complex (NPC). In some cases, drugpermeation of the nuclear membrane can be enhanced by selecting ananoparticle carrier with a size that allows penetration of the nuclearpores (<39 nm in diameter). In certain cases, drug permeation of thenuclear membrane can be enhanced by the addition of nuclear localizationsignal (NLS) peptides to the functional drug (gene carrier, DNA, etc.).This enhanced access to the nucleus can be achieved together with, orseparately from, a size of the nanoparticle carrier. If the charge onthe NLS peptide and the functional drug differ, however, drug deliverythrough the NPC may have limited success if the two oppositely chargedspecies combine and neutralize, as described in Akita et al.,Multilayered Nanoparticles for Penetrating the Endosome and NuclearMembrane via a Step-wise Membrane Fusion Process, Biomaterials, vol. 30(2009), pp. 2940-2049, which is hereby incorporated by reference hereinin its entirety. With a nanoparticle carrier, a multilayered structurecan be designed to separate the NLS peptide and an oppositely chargeddrug, thus enabling drug delivery into the nucleus of a cell with asingle nanoparticle. FIG. 19A shows a schematic view of a cross-sectionof nanoparticle carrier 230 with drug-loaded layer 232 including drug234, barrier layer 236, and NLS-agent-containing layer 238 including NLSagent 240. FIG. 19B shows a schematic view of a cross-section of ananoparticle carrier 230 with drug-loaded layer 232 and NLS-agentcontaining layer 238. In an example, drug-loaded layer 232 includes ananionic drug (e.g., an anionic plasmid DNA) and NLS-agent-containinglayer 236 includes a cationic NLS agent.

Further modifications and alternative embodiments of various aspectswill be apparent to those skilled in the art in view of thisdescription. Accordingly, this description is to be construed asillustrative only. It is to be understood that the forms shown anddescribed herein are to be taken as examples of embodiments. Elementsand materials may be substituted for those illustrated and describedherein, parts and processes may be reversed, and certain features may beutilized independently, all as would be apparent to one skilled in theart after having the benefit of this description. Changes may be made inthe elements described herein without departing from the spirit andscope as described in the following claims.

1. An imprint lithography method for forming nanoparticles comprising:forming a multilayer substrate comprising a base layer, a firstremovable layer bonded to the base layer, one or more functional layersbonded to the first removable layer; and a second removable layer bondedto one of the functional layers, wherein at least one of the one or morefunctional layers contains a functional material; forming a patternedlayer on a surface of the multilayer substrate in an imprint lithographyprocess, the patterned layer comprising a multiplicity of projectionsand recessions; transferring the features of the patterned layer intothe multilayer substrate in one or more etching processes to form aplurality of multilayer pillars extending from the base layer;dissolving the first and second removable layers with a solvent toseparate pillars containing the one or more functional layers from thebase layer, the separated pillars defining nanoparticles.
 2. The methodof claim 1 wherein the functional material is a pharmaceuticalcomposition.
 3. The method of claim 1 wherein the functional material isan imaging agent.
 4. The method of claim 1 further comprising forming ahard mask layer on the patterned layer, and then etching away at least aportion of the hard mask layer before transferring the features of thepatterned layer into the multilayer substrate in one or more etchingprocesses to form a plurality of multilayer pillars extending from thebase layer.
 5. The method of claim 1 further comprising forming a hardmask layer over the second removable layer.
 6. The method of claim 1wherein the transferring further substantially removes the patterninglayer.
 7. The method of claim 1 wherein at least one of the etchingprocesses comprises an inert gas etching process.
 8. The method of claim1 further comprising dissolving the second removable layer with a firstsolvent, wherein the first solvent selectively dissolves the secondremovable layer; and dissolving the first removable layer with a secondsolvent, wherein the second solvent selectively dissolves the firstremovable layer to separate the pillars from the base layer.
 9. Themethod of claim 1 further comprising treating the surfaces of themultilayer pillars before dissolving the first removable layer.
 10. Themethod of claim 9 wherein the surface treating further comprises coatingthe surface of the multilayer substrate.
 11. The method of claim 9wherein the surface treating further comprises modifying the surface ofthe multilayer substrate.
 12. The method of claim 11 wherein themodifying is selected from the group consisting of coupling ligands orother functional molecules to the surface, modifying surface charge, andmodifying the hydrophobicity or the hydrophilicity of the surface. 13.An imprint lithography method comprising: forming a multilayer substratecomprising a base layer, a first removable layer coupled to the baselayer, one or more functional layers coupled to the first removablelayer, and a second removable layer coupled to one of the functionallayers, wherein at least one of the functional layers comprises containsa functional material; forming a hard mask layer on the multilayersubstrate; forming a patterned layer over the hard mask layer in animprint lithography process, the patterned layer comprising amultiplicity of projections and recessions; transferring the features ofthe patterned layer into the hard mask and multilayer substrate in oneor more etching processes to form a plurality of multilayer pillarsextending from the base layer; and dissolving the first removable layerand the second removable layer with a solvent to separate the multilayerpillars from the base layer.
 14. The method of claim 13 wherein thefunctional material is a pharmaceutical composition.
 15. The method ofclaim 13 wherein the functional material is an imaging agent.
 16. Themethod of claim 13 wherein the transferring further substantiallyremoves the patterning layer and the hard mask.
 17. The method of claim13 further comprising dissolving the second removable layer with a firstsolvent, wherein the first solvent selectively dissolves the secondremovable layer; and dissolving the first removable layer with a secondsolvent, wherein the second solvent selectively dissolves the firstremovable layer to separate the pillars from the base layer.
 18. Aparticle formed by an imprint lithography process, the particlecomprising: at least a first layer and a second layer, the second layeradhered to the first layer, and wherein at least one of the first andsecond layers comprises a pharmaceutical composition or an imaging agentand a dimension of the particle is about 100 nm or less.
 19. Theparticle of claim 18 wherein the interface between the first and secondlayers is substantially planar.
 20. The particle of claim 18 furthercomprising at least a third layer adhered to the second layer, whereinthe second layer comprises a pharmaceutical composition and whereinneither the first nor the third layers comprises a pharmaceuticalcomposition.